ABSTRACT: OVERALL The goal of this proposal is to form a Biomedical Technology Research Resource (BTRR) at SLAC National Accelerator Laboratory that involves a set of interrelated Technology Research and Development (TR&D) projects aimed at enhancing and developing the unique capabilities of the SLAC Linac Coherent Light Source (LCLS) for biomedical applications. The BTRR will enable structural biology experiments that are extremely difficult or impossible to perform at synchrotron (SR) or electron microscopy (cryoEM) facilities and will increase the availability of these capabilities to the broader structural biology community. The enabled experiments will facilitate paradigm-shifting advances on a wide variety of topics, including neurotransmission, signal transduction, cellular metabolism, transcription and viral infection. The proposed TR&Ds are tightly coupled with the research themes of the nine Driving Biomedical Projects (DBPs). These research themes focus on developments to visualize large complexes and membrane proteins, such as GPCRs and that provide accurate active site structures of metalloenzymes, such as ribonucleotide reductase and cytochrome c oxidase, and complex macromolecular machines, such as RNA polymerase-II. Finally, a common research area of all DBPs involve time-resolved (TR) studies that include research to follow dynamic processes involved in adenine riboswitch signaling, the transport mechanism of N. gonorrhoeae MtrF, antibiotic binding to β-lactamase and examination of interaction specificity of CypA variants. All DBPs hinge on highly efficient data collection methods, which are required for successful macromolecular crystallography (MC) experiments at X-ray FELs. The high peak brightness of an X-ray FEL pulse destroys the crystal volume exposed, bringing about sample refreshment challenges previously unknown to the MC SR community. As a result, the sample must be continually replenished throughout the experiment. As part of the TR&Ds, sample injectors that rapidly deliver crystals and sample solutions to the X-ray beam will be optimized and automated during LCLS experiments along with data analysis to gauge experimental success and optimize use of limited sample and beam time. Time resolved studies hinge on improvements to mixing injectors, laser activation and complementary spectroscopic methods. X-ray FEL beam time is scarce so careful characterization of samples and complex experimental setups prior to beam time is critical to ensure experimental success, in particular for complex time resolved measurements of sensitive metalloenzymes intermediates. Experimental design and testing, sample production, sample characterization (including spectroscopic analysis) and crystal quality screening are supported in the laboratory, at the Stanford Synchrotron Radiation Lightsource (SSRL) and during screening beam time at LCLS. Integrating with, and enhancing the existing programs at SSRL and LCLS, the BTRR will provide support, expertise and training to the biomedical community.

NARRATIVE: OVERALL The proposal aims to develop new technologies to fully utilize the unique capabilities of the World’s first X-ray laser to understand how the molecules of life are assembled and how they move. The new technologies will provide high quality images and movies of molecules, allowing to understand molecules work with or against each other. The project will work with experts in the fields of drug design and enzymology, to name a few, to design and build new capabilities that will be made available to all scientists.